Parkinson's disease (PD) is characterized by a loss of dopamine (DA) neurons in substantia nigra and an accumulation of a-synuclein in Lewy bodies, the cytoplasmic inclusions of PD. These and other observations suggest that an understanding of the role of a-synuclein in these processes will provide insights into the pathogenesis of PD. Although the function of this protein is unknown, a-synuclein is likely to be a chaperone. This is suggested by a-synuclein's native unfolded structure, its ability to interact with several key cellular proteins, and its homology to 14-3-3, a family of molecular chaperones. One of the proteins to which 14-3-3 binds is tyrosine hydroxylase (TB), the rate limiting enzyme in catecholamine synthesis. The binding of 14-3-3 to TH occurs primarily to a phosphorylated serine (Ser19) in the N-terminal regulatory domain of the enzyme. The binding of 14-3-3 to Ser19 is required to increase the enzyme's activity and this activation correlates with an increase in catecholamine synthesis. An interaction of 14-3-3 with Ser40 on TH has recently been reported. We have preliminary data suggesting that: (1) a-synuclein and TB interact with each other in brain and in a dopaminergic cell line, (2) the addition of recombinant a-synuclein inhibits TH activity in an in vitro model of TH activity, and (3) overexpression of a-synuclein in a dopaminergic cell line inhibits TH activity, TH phosphorylation, and DA synthesis. Together these data suggest a key role for a-synuclein in TB regulation and link a-synuclein with the regulation of DA synthesis, storage, and release. Thus, the overall goal of this proposal is to explore the role of a-synuclein in TH regulation and DA synthesis and its impact on dopaminergic cell function under normal and pathophysiological conditions. To achieve this goal we propose three specific aims: (1) To test the hypothesis that a-synuclein inhibition of TH activity is associated with TH Ser19 and Ser40 phosphorylation.(2) To test the hypothesis that a-synuclein-induced TH-inhibition reduces DA release and that the absence of a-synuclein increases DA synthesis and release.(3) To test the hypothesis that a loss of a-synuclein-mediated inhibition of TH is associated with ROS formation and reduced cell viability. Using a unique combination of in vitro and in vivo approaches, our studies hold promise to expand both our basic understanding of TH regulation and provide further insight into the association between a-synuclein and TH as they relate to several abnormal conditions, including PD.